This application relates to a novel use of cancer markers such as kallikreins, including prostate-specific antigen (PSA), as inhibitors of angiogenesis useful for treating angiogenesis-related diseases such as angiogenesis-dependent cancer. The invention further relates to novel compositions and methods for curing angiogenesis-dependent cancer. In addition, the present invention relates to molecular probes for monitoring biosynthesis, to antibodies that are specific for serine proteases including kallikreins, to the development of peptide agonists and antagonists to kallikrein receptors, and to cytotoxic agents linked to kallikrein peptides.
Cancer Markers
The discovery of cancer markers and tumor markers has significantly enhanced not only diagnosis of cancer but has also contributed to the monitoring of cancer patients for assessing disease progression. A rise in cancer markers is a yardstick with which benign diseases can be distinguished from metastatic disease and also used to evaluate the efficacy of treatments. A decline in cancer markers is often a predictor of possible residual disease if the timing of blood sampling is soon after therapy. Numerous cancer markers are known in the art and are utilized in detection assays such as immunoassays depending upon the intrinsic characteristics of each marker (antigen specificity, molecular heterogeneity) and individual factors (nonspecific increases and renal and hepatic diseases).
Kallikrein
Kallikrein and kallikrein-like enzymes belong to a multigene family of serine proteases present in tissues and body fluids of numerous animals such as mammals and reptiles (i.e. snake venom). Included in the kallikrein family is hk1, a pancreatic/renal kallikrein; hk2, a human glandular kallikrein present in seminal fluid, a protease that activates urokinase type plasminogen activator; and prostate-specific antigen (hk3), a single-chain glycoprotein found in prostate tissue. Pre-kallikrein is converted by limited proteolysis into an active serine protease, and is one of the five major proteins involved in the activation and inhibition of surface mediated pathways in blood clotting. Pre-kallikrein is an important component of the biochemical junctures of intrinsic coagulation with other plasma proteolytic pathways required in the initiation, amplification, and propogation of surface-mediated defense reactions wherein various proteins such as bradykinin are involved. Thus, the molecular events of the contact phase of coagulation activation and inhibition involve pre-kallikrein and the plasma biochemical systems. (Colman et al. 1987).
Plasma kallikrein circulates in the blood as the precursor xe2x80x9cpre-kallikrein.xe2x80x9d Plasma pre-kallikrein is synthesized in the liver and secreted into plasma. However, only 25% of the protein exists as free pre-kallikrein and approximately 75% circulates bound to high molecular weight kininogen (HMWK). The molecular weight of human plasma pre-kallikrein, as assessed by gel filtration, is approximately 100,000 Daltons. By SDS polyacrylamide gel electrophoresis, plasma pre-kallikrein consists of two components having molecular weight 85,000 Daltons and 88,000 Daltons, depending whether the sample has undergone reduction. In plasma, the concentration of pre-kallikrein is estimated to be 35 xcexcg to 50 xcexcg/ml.
Following proteolysis, pre-kallikrein is activated to kallikrein and current studies do not demonstrate any clear cut difference in physiochemical or immunochemical properties of zymogen pre-kallikrein, and active enzyme kallikrein in the absence of reduction. Hageman factor (also known as Factor XIIa), and Hageman factor fragment (also known as Factor XIIf), are both able to convert pre-kallikrein to kallikrein. Unlike pre-kallikrein on reduced SDS gel electrophoresis, kallikrein has two types of subunits: a heavy chain with a molecular weight of approximately 52,000 Daltons, and two light chain variants with a molecular weight of approximately 36,000 Daltons and 33,000 Daltons. Pre-kallikrein circulates mostly complexed to high molecular weight kininogen HMWK, and it is thought that this complex may have protective functions for the pre-kallikrein. Following activation from pre-kallikrein to kallikrein, HMWK is cleaved to release bradykinin. Bradykinin is one of the most potent vasodilators known. (Colman et al. p.254).
The gene for plasma pre-kallikrein has not been isolated or characterized thus far. The messenger RNA for plasma pre-kallikrein, however has been characterized as a cDNA and shown to be approximately 2,300 nucleotides in length. It codes for a leader sequence of 19 amino acids and a mature polypeptide chain of 619 amino acids. The latter peptide in plasma pre-kallikrein is one amino acid longer than that in Factor XI. The activation reaction of pre-kallikrein to kallikrein is due to the cleavage of the peptide bond following arginine 371. Plasma kallikrein is generated as an enzyme composed of a heavy chain (371 amino acids) and a light chain (248 amino acids), held together by a disulfide bond. The catalytic domain or light chain of plasma kallikrein, contains three important amino acids (His-44, Asp-93 and Ser-188) that are directly involved in catalysis. In addition, plasma kallikrein contains 5 N-linked carbohydrate chains as established by amino acid sequence analysis.
The proteins and enzymes of the clotting cascade may perform multiple functions, for example, Factor XIIa may cleave pre-kallikrein to kallikrein, and Factor XI to XIa. Kallikrein can initiate reciprocal activation, generating additional Factor XIIa from Factor XII. Plasma kallikrein leads to the conversion of plasminogen to plasmin and Factor XIIa also converts plasminogen to plasmin. Kallikrein cleavage of HMWK results in the release bradykinin and may also elevate blood pressure by directly converting pro-renin to renin.
Alteration of any of the components of the vascular system, namely vessel cell wall, plasma proteins and platelets can result in an angiogenic disorder. There appear to be two major mechanisms under which the multiple inciting etilogies can be catagorized: endothelial injury and tissue injury. Endothelial injury relates to disease states such as infections that specifically injure the endothelium, with resultant kallikrein-kinin activation.
Injury to the vascular endothelium, such as occurs in endotoxemia, exposes basement membrane. Consequently collagen, along or in combination with proteoglycans or other components, activates Factor XII. Following Factor XII activation, intrinsic coagulation, activation of fibrinolysis and kinin formation occur. (Colman et al. p. 976).
Patients with bacterial infections, especially those caused by gram negative bacteria, may have elevated levels of plasma kallikrein. The hypotensive effect of kallikrein may contribute to the development of disseminated intravascular coagulation by reducing blood flow to reticuloendothelial organs thereby impairing clearance of activated coagulation factors.
Prostate-Specific Antigen
One important member of the kallikrein family is prostate-specific antigen. (Riegman et al.) The prostate-specific antigen (PSA) molecule is a single-chain glycoprotein consisting of approximately 237 amino acids and has a molecular weight of 28,430 daltons as determined by ion-spray mass spectroscopy. (Sokoll et al. 1997). The gene for PSA is located on the long arm of chromosome 19 and is approximately 6 kilobases in size, consisting of 4 introns and 5 exons. The PSA gene is under androgen regulation as evidenced by an androgen-responsive element in the promoter region. PSA is thought to be translated as a 261 amino acid prepropeptide. Although not isolated, the 244 propeptide zymogen form of PSA results after cleavage of the leader peptide during translation. The 237 amino acid active enzyme then is surmised to result from subsequent cleavage with as yet unidentified proteases. Structurally, the molecule is thought to possess five disulfide bonds owing to the presence of 10 cysteine residues with the active site of the enzyme composed of three amino acids, histidine 41, aspartate 96 and serine 189.
PSA is synthesized in the ductal epithelium and prostatic acini and located within the cell in cytoplasmic granules and vesicles, rough endoplasmic reticulum, vacuoles and secretory granules, and lysosmal dense bodies. PSA is found in normal hyperplastic, primary, and metatstatic prostate tissue. PSA is secreted into the lumina of the prostatic ducts via exocytosis to become a component of seminal plasma and reaches serum after diffusion from luminal cells through the epithelial basement membrane and prostatic stroma, where it can pass through the capillary basement membrane and epithelial cells or into the lymphatics. (Sokoll et al. 1997).
Despite original assumptions that PSA was a tissue-specific and gender-specific antigen, immunohistochemical and immunoassay methods have detected PSA in female and male periurethral glands, anal glands, apocrine sweat glands, apocrine breast cancers, salivary gland neoplasms, and most recently in human breast milk.
PSA functions as a serine protease exhibiting proteolytic activity similar to chymotrypsin, cleaving peptide bonds carboxy terminus of certain leucine and tyrosine residues. Based on its function, amino-acid structure and gene location, PSA is recognized as a member of the human kallikrein family.
In males, PSA is secreted from the lumen of the prostate and enters the seminal fluid as it passes through the prostate. In the seminal fluid are gel-forming proteins, primarily semenogelin I and II and fibronectin, which are produced in the seminal vesicles. These proteins are the major constituents of the seminal coagulum that forms at ejaculation and functions to entrap spermatozoa. PSA functions to liquefy the coagulum and break down the seminal clot through proteolysis of the gel-forming proteins into smaller more soluble fragments, thus releasing the spermatozoa. PSA also may modulate cell growth factor (IGF) binding protein 3, resulting in decreased binding with IGF-1, thus promoting cell growth. (Sokoll et al. 1997).
As it is used hereinafter, the term xe2x80x9cPSAxe2x80x9d refers to PSA as described above, peptide fragments of PSA that have angiogenesis inhibiting activity, and analogs of PSA that have substantial sequence homology (as defined herein) to the amino acid sequence of PSA, which have angiogenesis inhibiting activity.
Angiogenesis and Cancer
Several lines of direct evidence now suggest that angiogenesis is essential for the growth and persistence of solid tumors and their metastases (Folkman, 1989; Hori et al., 1991; Kim et al., 1993; Millauer et al., 1994). To stimulate angiogenesis, tumors upregulate their production of a variety of angiogenic factors, including the fibroblast growth factors (FGF and bFGF) (Kandel et al., 1991) and vascular endothelial cell growth factor/vascular permeability factor (VEGF/VPF). However, many malignant tumors also generate inhibitors of angiogenesis, including ANGIOSTATIN(copyright) protein and thrombospondin. (Chen et al., 1995; Good et al., 1990; O""Reilly et al., 1994). It is postulated that the angiogenic phenotype is the result of a net balance between these positive and negative regulators of neovascularization. (Good et al., 1990; O""Reilly et al., 1994; Parangi et al., 1996; Rastinejad et al., 1989). Several other endogenous inhibitors of angiogenesis have been identified, although not all are associated with the presence of a tumor. These include, platelet factor 4 (Gupta et al., 1995; Maione et al., 1990), interferon-alpha, interferon-inducible protein 10 (Angiolillo et al., 1995; Strieter et al., 1995), which is induced by interleukin-12 and/or interferon-gamma (Voest et al., 1995), gro-beta (Cao et al., 1995), and the 16 kDa N-terminal fragment of prolactin (Clapp et al., 1993).
One example of an angiogenesis inhibitor that specifically inhibits endothelial cell proliferation is ANGIOSTATIN(copyright) protein. (O""Reilly et al., 1994). ANGIOSTATIN(copyright) protein is an approximately 38 kiloDalton (kDa) specific inhibitor of endothelial cell proliferation. ANGIOSTATIN(copyright) protein is an internal fragment of plasminogen containing at least three of the five kringles of plasminogen ANGIOSTATIN(copyright) protein has been shown to reduce tumor weight and to inhibit metastasis in certain tumor models. (O""Reilly et al., 1994). Another angiogenesis inhibitor is ENDOSTATIN(copyright) protein, which is a carboxy fragment of collagen XV or XVIII. (O""Reilly et al., 1997).
What is needed is the discovery and development of additional anti-angiogenic agents that may be used alone or in combination with known angiogenic agents in order to treat cancer and hyperproliferative disorders.
The present invention generally relates to cancer markers including kallikreins and specifically to prostate-specific antigen (PSA) as angiogenesis inhibitors and methods of use thereof. PSA is a potent and specific inhibitor of endothelial cell function and angiogenesis. Systemic therapy with kallikreins such as PSA, causes suppression of tumor-induced angiogenesis, and exhibits strong antitumor activity.
PSA has a molecular weight of approximately 28,430 Daltons as determined by ion-spray mass spectroscopy and is capable of inhibiting endothelial cell function in cultured endothelial cells.
The present invention provides methods and compositions for treating diseases and processes mediated by undesired and uncontrolled angiogenesis by administering to a human or animal with the undesired angiogenesis a composition comprising serine proteases including kallikreins such as purified PSA, or PSA derivatives, in a dosage sufficient to inhibit angiogenesis. The present invention is particularly useful for treating or for repressing the growth of tumors. Administration of PSA to a human or animal with metastasized tumors prevents the growth or expansion of those tumors. The invention further provides methods and compositions for regulating endothelial cell function in vivo as well as in vitro.
The present invention also includes kallikrein peptide fragments that can be labeled isotopically or with other molecules or proteins for use in the detection and visualization of kallikrein binding sites with state of the art techniques, including, but not limited to, positron emission tomography, autoradiography, flow cytometry, radioreceptor binding assays, and immunohistochemistry.
The present invention also includes PSA, PSA fragments, or PSA receptor agonists and antagonists linked to cytotoxic agents for therapeutic and research applications.
In addition, PSA peptides may act as agonists and antagonists of the PSA receptor, thereby enhancing or blocking the biological activity of PSA. Such peptides are used in the isolation of the PSA receptor.
A surprising discovery is that various forms of serine proteases including recombinant kallikreins, such as recombinant PSA proteins, can serve as sustained release anti-angiogenesis compounds when administered to a tumor-bearing animal.
The present invention also relates to methods of using PSA protein and peptide fragments, corresponding nucleic acid sequences, and antibodies that bind specifically to the inhibitor and its peptides, to diagnose endothelial cell-related diseases and disorders.
The invention further encompasses a method for identifying receptors specific for PSA, and the receptor molecules identified and isolated thereby.
An important medical method is a new form of birth control, wherein an effective amount of kallikrein (for example PSA) is administered to a female such that uterine endometrial vascularization is inhibited and embryo implantation cannot occur or be sustained.
A particularly important aspect of the present invention is the discovery of a novel and effective method for treating angiogenesis-related diseases, particularly angiogenesis-dependent cancer, in patients, and for curing angiogenesis-dependent cancer in patients. The method unexpectedly provides the medically important result of inhibition of tumor growth and reduction of tumor mass. The method relates to the co-administration of a serine protease or kallikrein of the present invention and another anti-angiogenesis compound, such as ENDOSTATIN(copyright) protein or ANGIOSTATIN(copyright) protein. Accordingly, the present invention also includes formulations containing PSA, ENDOSTATIN(copyright) protein, and/or ANGIOSTATIN(copyright) protein, which are effective for treating or curing angiogenesis-dependent diseases.
Accordingly, it is an object of the present invention to provide compositions and methods comprising serine proteases including kallikreins useful for the treatment of angiogenic disorders.
Another object of the present invention is to provide compositions and methods comprising prostate-specific antigen useful for the treatment of angiogenic disorders.
It is another object of the present invention to provide compositions and methods of treating diseases and processes that are mediated by angiogenesis.
It is yet another object of the present invention to provide compositions and methods for treating diseases and processes that are mediated by angiogenesis including, but not limited to, hemangioma, solid tumors, leukemia, metastasis, telangiectasia psoriasis scleroderma, pyogenic granuloma, myocardial angiogenesis, plaque neovascularization, coronary collaterals, cerebral collaterals, arteriovenous malformations, ischemic limb angiogenesis, corneal diseases, rubeosis, neovascular glaucoma, diabetic retinopathy, retrolental fibroplasia, arthritis, diabetic neovascularization, macular degeneration, wound healing, surgical adhesions, peptic ulcer, fractures, keloids, vasculogenesis, hematopoiesis, ovulation, menstruation, and placentation.
It is another object of the present invention to provide compositions and methods for treating or repressing the growth of a cancer.
Still another object of the present invention is to provide compositions and methods consisting of antibodies to PSA that are selective for specific regions of the PSA molecule.
It is another object of the present invention to provide compositions and methods for the detection or prognosis of anti-angiogenesis activity.
It is yet another object of the present invention to provide a therapy for cancer that has minimal side effects.
Still another object of the present invention is to provide compositions comprising PSA or PSA peptide linked to a cytotoxic agent for treating or repressing the growth of a cancer.
These and other objects, features and advantages of the present invention will become apparent after a review of the following detailed description of the disclosed embodiments and the appended claims.